Optimal Conditions for Biomass and Recombinant Glycerol Kinase Production Using the Yeast Pichia pastoris

The extracellular glycerol kinase gene from Saccharomyces cerevisiae (GUT]) was cloned into the expression vector pPICZ alpha. A and integrated into the genome of the methylotrophic yeast Pichia pastoris X-33. The presence of the GUT1 insert was confirmed by PCR analysis. Four clones were selected and the functionality of the recombinant enzyme was assayed. Among the tested clones, one exhibited glycerol kinase activity of 0.32 U/mL, with specific activity of 0.025 U/mg of protein. A medium optimized for maximum biomass production by recombinant Pichia pastoris in shaker cultures was initially explored, using 2.31 % (by volume) glycerol as the carbon source. Optimization was carried out by response surface methodology (RSM). In preliminary experiments, following a Plackett-Burman design, glycerol volume fraction (phi(Gly)) and growth time (t) were selected as the most important factors in biomass production. Therefore, subsequent experiments, carried out to optimize biomass production, followed a central composite rotatable design as a function of phi(Gly) and time. Glycerol volume fraction proved to have a significant positive linear effect on biomass production. Also, time was a significant factor (at linear positive and quadratic levels) in biomass production. Experimental data were well fitted by a convex surface representing a second order polynomial model, in which biomass is a function of both factors (R(2)=0.946). Yield and specific activity of glycerol kinase were mainly affected by the additions of glycerol and methanol to the medium. The optimized medium composition for enzyme production was: 1 % yeast extract, 1 % peptone, 100 mM potassium phosphate buffer, pH=6.0, 1.34 % yeast nitrogen base (YNB), 4.10(-5) % biotin, 1 %, methanol and 1 %, glycerol, reaching 0.89 U/mL of glycerol kinase activity and 14.55 g/L of total protein in the medium after 48 h of growth.

Optimisation of conditions for the extraction of casearins from Casearia sylvestris using response surface methodology

Optimal conditions for the extraction of casearins from Casearia sylvestris were determined using response surface methodology. The maceration and sonication extraction techniques were performed using a 3 x 3 x 3 full factorial design including three acidity conditions, three solvents of different polarities and three extraction times. The yields and selectivities of the extraction of casearins were significantly influenced by acidity conditions. Taking into account all variables tested, the optimal conditions for maceration extraction were estimated to involve treatment with dichloromethane saturated with ammonium hydroxide for 26 h. Similar yields and selectivities for casearins were determined for sonication extraction using the same solvent but for the much shorter time of I h. The best results for stabilisation of the fresh plant material were obtained using leaves that had been oven dried at 40 degrees C for 48 h. Copyright (c) 2006 John Wiley & Sons, Ltd.

Computed tomographic-dacryocystography (CT-DCG) of the normal canine nasolacrimal drainage system with three-dimensional reconstruction

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro Survival of Follicles Collected from Domestic Cats' Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Setting the preheating and steady-state operation of electronic ballasts, considering electrodes of hot-cathode fluorescent lamps

This paper presents a new methodology for the adjustment of the preheating process and steady-state operation of electronic ballasts intended for hot-cathode fluorescent lamps. The classical series-resonant parallel-loaded half-bridge inverter is the power stage analyzed in this paper. In addition, the preheating process is based on the imposition of a constant rms current through the electrodes, in order to provide a proper value of the R-h/R-c ratio before the lamp start. According to the proposed methodology, it is possible to set suitable operating points for, the electronic ballast, considering optimal conditions for the lamps electrodes. Therefore, the proposed methodology for setting the preheating and steady-state operation is a complete platform to the design of electronic ballasts for hot-cathode fluorescent lamps.

FAAS determination of metal nutrients in fish feed after ultrasound extraction

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.

Xylanolytic complex from Aspergillus giganteus: production and characterization

An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid VOGEL medium containing xylan as carbon source, pH 6.5 to 7.0, at 25degreesC and. under shaking at 120 rpm during 84h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar-cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50degreesC and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T-50 of 2 h, 13 min and I min when it was incubated at 40, 50 and 60degreesC, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg+2, Cu+2 and SDS at 10 mm. The analysis of the products from the oat spelts xylan hydrolysis through thin-layer chromatography indicated endoxylanase activity, lack of debranching enzymes and P-xylosidase activity in assay conditions.

Xylanase production by Aspergillus versicolor

The xylanolytic system of Aspergillus versicolor is controlled by induction and carbon catabolite repression. Carboxymethylcellulose and wheat bran were the best inducers of xylanolytic activity. When the fungus was grown for 5 days on VOGEL's liquid medium with wheat bran, the optimal pH and temperature for xylanase production were 6.5 and 30 degrees C, respectively. Optimal conditions for the xylanolytic activity assay were at pH 6.0 and 55 degrees C. The half-life at 60 degrees C of the crude enzyme was 6.5 and 21 minutes, in the absence or presence of substrate, respectively.Xylan is the main hemicellulosic component of plant biomass being present in appreciable quantities in agricultural and several agroindustrial wastes. From the products of xylan enzymatic hydrolysis it is possible to obtain cell protein, fuels and other chemicals. Xylanases combined with cellulase could have applications in food processing. Cellulase-free xylanases can be also utilized for preparation of cellulose pulps and liberation of textile fibres (WOODWARD 1984; BIELY 1985, WONG et al. 1988). In view of the potential applications of xylanases, a study of these enzymes from various sources and their multiplicity is desirable.Among xylanolytic microorganisms, filamentous fungi have been more extensively studied and the genus Aspergillus has been shown to be an efficient producer of xylanases. Preliminary observations from our laboratory have demonstrated that a strain of Aspergillus versicolor, isolated from Brazilian soil, produced high xylanase and low cellulase levels, which is an interesting characteristic for some industrial applications. In this report we describe the production and some properties of xylanase obtained from this fungus.

Production and characterization of cellulase-free xylanase from Trichoderma inhamatum

The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25 degrees C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50 degrees C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T(1/2) was 2.2 h at 40 degrees C and 2 min at 50 degrees C. The pH stability was high from 4.0 to 11.0.These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence.

Utilização do resíduo líquido de indústria de processamento de suco de laranja como meio de cultura de Penicillium citrinum: depuração biológica do resíduo e produção de enzima

Penicillium citrinum grown in orange juice processing wastes medium under continuous agitation was studied in order to establish optimal conditions (incubation period, incubation temperature, initial pH and nitrogen addition) for biomass and ribonuclease production, as well as, biological depuration of the wastes. Nitrogen addition to wastes medium increased the biomass and ribonuclease production and provides COD reduction. The soy meal shows to be the best nitrogen source. The conditions for a more favorable enzyme and biomass production and COD reduction were initial pH 6.0 and temperature of 27°C. The maximum value obtained for these parameters on optimal conditions of cultivation was 11 U/mL of enzyme, 4 mg/mL of biomass (dry matter) and 55% of COD reduction, in 96 hours of incubation.

Esterase inhibition in tadpoles of Scinax fuscovarius (Anura, Hylidae) as a biomarker for exposure to organophosphate pesticides

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A simple and green analytical method for determination of glyphosate in commercial formulations and water by diffuse reflectance spectroscopy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

An environmentally friendly reflectometric method for ranitidine determination in pharmaceuticals and human urine

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Determinação espectrofotométrica por injeção em fluxo de glifosato em formulações comerciais de herbicidas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)